Acidification of a wide array of intracellular organelles, including clathrin-coated vesicles, lysosomes, chromaffin granules and other endomembranes has been shown to be mediated by a new class of vacuolar- type proton translocating ATPases. In addition, renal acidification and osteoclast-mediated bone reabsorption are controlled by vacuolar-type proton pumps. It is the central aim of this proposal to define the molecular characteristics of a prototype of this class of H+ ATPases, the clathrin-coated vesicle proton pump. This enzyme, like all vacuolar-type H+ ATPases, is a large (>500 kDa) hetero-oligomer, composed of 8-9 different subunits. Recently, an important sector of the enzyme, the ATP hydrolytic center, has undergone partial elucidation. Several key issues are detailed in this proposal, including definition of the roles of four subunits (70, 58, 40 and 33 kDa) in ATP hydrolysis, the role of a 116 kDa component in pump function, and the characterization of a novel activator of the pump. Definition of subunit function will be based upon the reconstitution of ATPase activity through the use of recombinant subunits. In addition, the recombinant 116 kDa component will be reconstituted to biochemically-prepared subcomplexes to determine its function. Characterization of the activator of the pump will include its molecular cloning, expression, and investigation of its mechanism of pump regulation. Delineation of the molecular characteristics of the clathrin-coated vesicle proton pump will provide the groundwork necessary for future studies of the regulation and biogenesis of this multimeric enzyme.